Thrombin peptide derivative dimers

ABSTRACT

Disclosed are thrombin peptide derivative dimers comprising two polypeptides having the amino acid sequence SEQ ID NO. 2: Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val, or a C-terminal truncated fragment of the polypeptide having at least six amino acids. Zero, one, two, or three amino acids in the polypeptide or polypeptide fragment differ from the corresponding position of SEQ ID NO. 2. Also disclosed are methods of treating a subject in need of treatment with a thrombin receptor agonist. The methods comprise the step of administering an effective amount of the thrombin peptide derivative described above.

RELATED APPLICATION(S)

This application is a continuation of International Application No.PCT/US2003/020626, which designated the United States and was filed Jul.1, 2003, published in English, which claims the benefit of U.S.Provisional Application No. 60/393,579, filed Jul. 2, 2002. The entireteachings of the above applications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Thrombin, a multi-functional enzyme already known for its blood-clottingactivity, has been recently reported to be an important cell-growthfactor. For example, thrombin has been shown to promote angiogenesis,the development of new blood vessels, and to stimulate endothelial cellproliferation. These processes are a pivotal part of healing wounds.

Thrombin peptide derivatives are molecules having an amino acid sequencederived at least in part from that of thrombin, which are active atcertain thrombin receptors. For example, thrombin peptide derivativesfrom amino acids 508-530 of human pro-thrombin have been described bythe present inventors for promoting thrombin receptor mediated cellstimulation and for their use in the treatment of wounds, andstimulation of angiogenesis (see, e.g., U.S. Pat. No. 5,500,412 or5,352,664, the contents of which are incorporated herein by reference intheir entirety). Because of their biological activity, these thrombinpeptide derivatives show great potential as pharmaceuticals. TP508 isone such example of a thrombin peptide derivative and has the amino acidsequence ofH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO. 1).

Strict regulations by the Food and Drug Administration (FDA) require ahigh degree of purity of biologically active agents when used aspharmaceuticals. It therefore is necessary to obtain active thrombinpeptide derivatives that maintain their purity over extended timeperiods, if these compounds are to be used to treat humans. For example,the purity of TP508 diminishes over time because of dimerization. Forexample, TP508 has a half-life of about 2 to about 4 hours in bufferedsolutions at neutral pH.

SUMMARY OF THE INVENTION

It has now been found that thrombin peptide derivative dimers retainactivity toward thrombin receptors. Thrombin peptide derivative dimerscan be prepared essentially free of monomer and have about the samelevel of activity toward the thrombin receptor as TP508 (see Example 3).The thrombin peptide derivative dimers also retain their purity withminimal reversion to monomer (see Example 2). Based on this discovery,the invention provides novel peptide dimers, pharmaceutical compositionscomprising these peptide dimers, and methods useful for treating asubject in need of treatment with a thrombin receptor agonist.

One embodiment of the present invention is a thrombin peptide derivativedimer comprising two thrombin peptide derivatives. Each thrombin peptidederivative independently comprises a polypeptide which has the aminoacid sequence of SEQ ID NO. 2:Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val, or a C-terminaltruncated fragment thereof having at least six amino acids. Zero, one,two, or three amino acids in the polypeptide differ from thecorresponding position of SEQ ID NO. 2. Preferably the difference isconservative. The thrombin peptide derivatives are optionally amidatedat the C-terminus and/or acylated at the N-terminus.

Another embodiment of the invention also relates to pharmaceuticalcompositions comprising a thrombin receptor agonist or a thrombinpeptide derivative dimer described herein and a pharmaceuticallyacceptable carrier or diluent.

Another embodiment of the invention further relates to methods oftreating a subject in need of treatment with a thrombin receptoragonist. The methods comprise the step of administering an effectiveamount of a thrombin peptide derivative dimer described herein.

Advantages of the thrombin peptide derivative dimers of the presentinvention include longer storage life in solution compared with themonomer TP508. Therefore, it is possible to deliver precise andreproducible dosages with the disclosed peptides, even after storage forprolonged periods of time. The thrombin peptide derivative dimersdescribed herein are also inexpensive to produce. The thrombin peptidederivative dimers can be used in the treatment and/or prevention ofdiseases and/or conditions in which angiogenesis and cell proliferationwould be beneficial. The thrombin peptide derivative dimers can be usedto help treat, for example, wounds such as diabetic ulcers, bonefractures, and cartilage damage. The thrombin peptide derivative dimerscan also be used to prevent restenosis in patients after angioplasty andregenerate blood vessels in cardiac tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the conversion of TP508 to dimer over time.The graph shows the HPLC peak area measurements of TP508-monomer,TP508-dimer and unknowns found in samples of TP508 saline solution (5mg/mL, incubated at 4° C.), taken at intervals over a time period of 6months. Peak area is indicated as percent. Time is indicated as days.Monomer is indicated as (-●-). Dimer is indicated as (...∘...). Unknownsare indicated as (--▾--).

FIG. 2 is a graph showing that the thrombin peptide dimer retains thebiological activity of TP508 with regard to the acceleration of woundhealing. The graph shows wound area measurements (indicated in mm²) onthe dorsum of male Sprague-Dawley rats from post-wounding Day 7 and Day10. The saline vehicle control is indicated as “vehicle,” the TP508control is indicated as “TP508,” low-dose thrombin peptide dimer isindicated as “lo-di” and high-dose thrombin peptide dimer is indicatedas “hi-di.”

DETAILED DESCRIPTION OF THE INVENTION

Applicants have found that the thrombin peptide derivative dimers of thepresent invention essentially do not revert to monomers and still haveabout the same biological activity as the thrombin peptide derivativesof the prior art. A “thrombin peptide derivative dimer” is a moleculecomprising two thrombin peptide derivatives linked by a covalent bond,preferably a disulfide bond between cysteine residues. Thrombin peptidederivative dimers are typically essentially free of the correspondingmonomer, e.g., greater than 95% free by weight and preferably greaterthan 99% free by weight. Preferably the polypeptides are the same andcovalently linked through a disulfide bond.

It will be understood that the thrombin peptide derivatives disclosedherein can have C-terminal amides. A “C-terminal amide” is an amide atthe C-terminal amino acid residue in which the alpha carboxylic acid isreplaced with an amide. For example, C-terminal amino acid amideresidues have the formula: —NH—CH(R_(a))—C(O)—NR_(b)R_(c). R_(a) is anamino acid side chain. An amino acid side chain can be hydrogen, asubstituted or unsubstituted C₁-C₁₀ aliphatic group, or a substituted orunsubstituted C₁-C₁₀ aromatic group. Preferably R_(a) is an amino acidside chain corresponding to naturally occurring amino acids. R_(b) andR_(c) are independently hydrogen, a C₁-C₁₀ substituted or unsubstitutedaliphatic group, or R_(b) and R_(c), taken together with the nitrogen towhich they are bonded, form a C₁-C₁₀ non-aromatic heterocyclic group.Preferably, the C-terminal amide is —C(O)NH₂ (carboxamide). As usedherein, “—NH₂” at the C-terminus indicates a C-terminus carboxamide;“—OH” at the C-terminus indicates that the peptide has a freeC-terminus; and no designation at the C-terminus indicates that thepeptide is amidated at the C-terminus or has a free C-terminus.

It will also be understood that the thrombin peptide derivativesdisclosed herein can have an acylated N-terminus. An “acylatedN-terminus” is an N-terminal amino acid residue in which the nitrogen ofthe N-terminal amino acid residue is acylated. For example, acylatedN-terminal amino acids residues have the formula:R_(d)C(O)—NH—CHR_(a)—C(O)—. R_(d) is hydrogen, a C₁-C₁₀ substituted orunsubstituted aliphatic group, or a C₁-C₁₀ substituted or unsubstitutedaromatic group. Acetyl is a preferred acyl group. An “—H” at theN-terminus indicates that the N-terminus is unsubstituted; and nodesignation at the N-terminus indicates that the terminus is acylated orunsubstituted.

Preferably, the N-terminus of a thrombin peptide derivative is free(i.e., unsubstituted) and the C-terminus is free (i.e., unsubstituted)or amidated, preferably a carboxamide (i.e., —C(O)NH₂).

Thrombin peptide derivatives are believed to activate cells by bindingto a high-affinity cell-surface thrombin receptor known as thenon-proteolytically-activated thrombin receptor (hereinafter “NPAR”) (R.Horvat, et. al., J. Cell Sci. 108, 1155-1164, 1995). Compounds whichstimulate NPAR are said to be thrombin receptor agonists. NPARactivation can be assayed based on the ability of molecules to stimulatecell proliferation when added to fibroblasts in the presence ofsubmitogenic concentrations of thrombin or molecules that activateprotein kinase C or compete with ¹²⁵I-thrombin for high affinity bindingto thrombin receptors, as disclosed in U.S. Pat. Nos. 5,352,664 and5,500,412 and in Glenn et al., J. Peptide Research 1: 65 (1988).

Thrombin peptide derivatives stimulate NPAR and have less than aboutfifty amino acids, preferably less than about thirty-three amino acids.Thrombin peptide derivatives also have sufficient homology to thefragment of human thrombin corresponding to prothrombin amino acids508-530:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 3) so that the polypeptide activates NPAR. The thrombinpeptide derivative dimers described herein are formed from polypeptidestypically having at least six amino acids and preferably between about12 and 33 amino acids, more preferably between about 12 and 23 aminoacids.

In a first preferred embodiment, each thrombin peptide derivativecomprises a polypeptide having the amino acid sequence of SEQ ID NO. 4:Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val, or a C-terminaltruncated fragment thereof having at least six amino acids. Morepreferably, each thrombin peptide derivative has the amino acid sequenceof SEQ ID NO. 5:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val,or a fragment thereof comprising amino acids 10-18 of SEQ ID NO. 5. Evenmore preferably, the thrombin peptide derivative has the amino acidsequence SEQ ID NO. 6:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X,-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val,or a fragment thereof comprising amino acids 10-18 of SEQ ID NO. 6. X₁is Glu or Gln and X₂ is Phe, Met, Leu, His or Val. Preferably X₁ is Glu,and X₂ is Phe. One example of a thrombin peptide derivative of this typeis a polypeptide having the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 3). A further example of a thrombin peptide derivative ofthis type is a polypeptide having the amino acid sequenceH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO. 1). Zero, one, two or three amino acids in the thrombinpeptide derivative differ from the amino acid at the correspondingposition of SEQ ID NO. 1, 3, 4, 5 or 6. Preferably, the difference isconservative.

One example of a thrombin peptide derivative dimer of the presentinvention is represented by Formula I:

In a second preferred embodiment, each thrombin peptide derivativecomprises a polypeptide having the amino acid sequence SEQ ID NO. 7:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr,or a C-terminal truncated fragment thereof having at least twenty-threeamino acids. More preferably, each thrombin peptide derivative has theamino acid sequence SEQ ID NO. 8:Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr,or a C-terminal truncated fragment thereof having at least twenty-threeamino acids. X₁ is Glu or Gln and X₂ is Phe, Met, Leu, His or Val.Preferably X₁ is Glu, and X₂ is Phe. One example of a thrombin peptidederivative of this type is a polypeptide having the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr(SEQ ID NO. 9). A further example of a thrombin peptide derivative ofthis type is a polypeptide having the amino acid sequenceH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr-NH₂(SEQ ID NO. 10). Zero, one, two or three amino acids in the thrombinpeptide derivative differ from the amino acid at the correspondingposition of SEQ ID NO. 7, 8, 9 or 10. Preferably, the difference isconservative.

A “conservative substitution” is the replacement of an amino acid withanother amino acid that has the same net electronic charge andapproximately the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have approximately the samesize when the total number carbon and heteroatoms in their side chainsdiffers by no more than about four. They have approximately the sameshape when the number of branches in the their side chains differs by nomore than one. Amino acids with phenyl or substituted phenyl groups intheir side chains are considered to have about the same size and shape.Listed below are five groups of amino acids. Replacing an amino acid ina polypeptide with another amino acid from the same group results in aconservative substitution:

-   -   Group I: glycine, alanine, valine, leucine, isoleucine, serine,        threonine, cysteine, and non-naturally occurring amino acids        with C1-C4 aliphatic or C1-C4 hydroxyl substituted aliphatic        side chains (straight chained or monobranched).    -   Group II: glutamic acid, aspartic acid and non-naturally        occurring amino acids with carboxylic acid substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group III: lysine, ornithine, arginine and non-naturally        occurring amino acids with amine or guanidino substituted C1-C4        aliphatic side chains (unbranched or one branch point).    -   Group IV: glutamine, asparagine and non-naturally occurring        amino acids with amide substituted C1-C4 aliphatic side chains        (unbranched or one branch point).    -   Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.

A “highly conservative substitution” is the replacement of an amino acidwith another amino acid that has the same functional group in the sidechain and nearly the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have nearly the same sizewhen the total number carbon and heteroatoms in their side chainsdiffers by no more than two. They have nearly the same shape when theyhave the same number of branches in the their side chains. Examples ofhighly conservative substitutions include valine for leucine, threoninefor serine, aspartic acid for glutamic acid and phenylglycine forphenylalanine. Examples of substitutions which are not highlyconservative include alanine for valine, alanine for serine and asparticacid for serine.

An “N-terminal truncated fragment” refers to a fragment remaining afterremoving an amino acid or block of amino acids from the N-terminus,preferably a block of no more than six amino acids, more preferably ablock of no more than three amino acids. Optionally, an N-terminaltruncated fragment is acylated and/or amidated as described above.

A “C-terminal truncated fragment” refers to a fragment remaining afterremoving an amino acid or block of amino acids from the C-terminus,preferably a block of no more than six amino acids, more preferably ablock of no more than three amino acids. Optionally, a C-terminaltruncated fragment is amidated and/or acylated as described above.

A “non-aromatic heterocyclic group”, as used herein, is a non-aromaticcarbocyclic ring system that has 3 to 10 atoms and includes at least oneheteroatom, such as nitrogen, oxygen, or sulfur. Examples ofnon-aromatic heterocyclic groups include piperazinyl, piperidinyl,pyrrolidinyl, morpholinyl and thiomorpholinyl.

The term “aryl group”, as used herein, includes both carbocyclic andheterocyclic aromatic ring systems. Examples of aryl groups includephenyl, indolyl, furanyl and imidazolyl.

An “aliphatic group” is a straight chain, branched or cyclicnon-aromatic hydrocarbon. An aliphatic group can be completely saturatedor contain one or more units of unsaturation (e.g., double and/or triplebonds), but is preferably saturated, i.e., an alkyl group. Typically, astraight chained or branched aliphatic group has from 1 to about 10carbon atoms, preferably from 1 to about 4, and a cyclic aliphatic grouphas from 3 to about 10 carbon atoms, preferably from 3 to about 8.Aliphatic groups include, for example, methyl, ethyl, n-propyl,iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, cyclopentyl, hexyl,cyclohexyl, octyl and cyclooctyl.

Suitable substituents for an aliphatic group, an aryl group or anon-aromatic heterocyclic group are those which do not significantlylower therapeutic activity of the thrombin peptide derivative, forexample, those found on naturally occurring amino acids. Examplesinclude —OH, a halogen (—Br, —Cl, —I and —F), —O(R_(e)), —O—CO—(R_(e)),—CN, —NO₂, —COOH, ═O, —NH₂—NH(R_(e)), —N(R_(e))₂, —COO(R_(e)), —CONH₂,—CONH(R_(e)), —CON(R_(e))₂, —SH, —S((R_(e))), an aliphatic group, anaryl group and a non-aromatic heterocyclic group. Each R_(e) isindependently an alkyl group or an aryl group. A substituted aliphaticgroup can have more than one substituent.

A “subject” is preferably a human, but can also be an animal in need oftreatment with a thrombin receptor agonist, e.g., companion animals(e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horsesand the like) and laboratory animals (e.g., rats, mice, guinea pigs andthe like).

Subjects “in need of treatment” with a thrombin receptor agonist, aresubjects with diseases and/or conditions that can be treated withthrombin receptor agonists and thrombin peptide derivative dimers toachieve a beneficial therapeutic and/or prophylactic result. Abeneficial outcome includes a decrease in the severity of symptoms ordelay in the onset of symptoms, increased longevity and/or more rapid ormore complete resolution of the disease or condition. For example, asubject in need of treatment requires cell proliferation involvingchondrocytes, angiogenesis, bone growth, cardiac repair, wound healingor inhibition of restenosis.

Thrombin peptide derivatives have been shown to stimulate proliferationof endothelial cells, fibroblasts, and keratinocytes (see, e.g., U.S.Pat. No. 5,500,412 or 5,352,664, the contents of which are incorporatedherein by reference in their entirety). The disclosed thrombin peptidederivative dimers can therefore be used to promote healing in acutewounds such as burns, dermal wounds, surgical wounds, and bonefractures. In addition, thrombin peptide derivatives have recently beenshown to be particularly effective in promoting the healing of chronicwounds such as, diabetic ulcers, venous ulcers, and pressure sores (see,e.g., WO 03/013569, the contents of which are incorporated herein byreference in their entirety). Thrombin peptide derivatives have alsobeen shown to stimulate the growth of chondrocytes (see, e.g., WO02/07748, the contents of which are incorporated herein by reference intheir entirety). Thus thrombin peptide derivatives, including thecompounds of the present invention can be used to stimulate chondrocytegrowth and repair in, for example patients with osteoarthritis or jointinjuries. Other uses for thrombin peptide derivatives, including thoseof the present invention, include stimulating bone growth to promotehealing of simple fractures, non-union fractures, voids and gaps in boneand bone grafts, preventing restenosis in patients after angioplasty andpromoting the regeneration of blood vessels in cardiac tissue (see,e.g., WO 02/005836 and WO 02/004008, the contents of which areincorporated herein by reference in their entirety).

An “effective amount” is the quantity of thrombin peptide derivativedimer that results in an improved clinical outcome of the conditionbeing treated with the thrombin peptide derivative dimer compared withthe absence of treatment. The amount of thrombin peptide derivativedimer administered will depend on the degree, severity, and type of thedisease or condition, the amount of therapy desired, and the releasecharacteristics of the pharmaceutical formulation. It will also dependon the subject's health, size, weight, age, sex and tolerance to drugs.Typically, the agonist is administered for a sufficient period of timeto achieve the desired therapeutic effect. Typically between about 1 μgper day and about 1 mg per day of the thrombin peptide derivative(preferably between about 5 μg per day and about 100 μg per day) isadministered to the subject in need of treatment.

The thrombin peptide derivative dimer can be administered by anysuitable route, locally or systemically, including, for example, byparenteral administration. Parenteral administration can include, forexample, intramuscular, intravenous, subcutaneous, or intraperitonealinjection. Topical administration for treating wounds can include, forexample, creams, gels, ointments or aerosols. Respiratory administrationcan include, for example, inhalation or intranasal drops. For certainindications such as stimulating bone growth, cartilage repair, cardiacrepair and the treatment of restenosis, it is advantageous to inject orimplant the thrombin peptide derivative directly to the treatment site.The thrombin peptide derivative dimer can also be advantageouslyadministered in a sustained release formulation.

The thrombin peptide derivative dimer can be administered to the subjectin conjunction with an acceptable pharmaceutical carrier as part of apharmaceutical composition. The formulation of the pharmaceuticalcomposition will vary according to the route of administration selected.Suitable pharmaceutical carriers may contain inert ingredients which donot interact with the compound. The carriers should be biocompatible,i.e., non-toxic, non-inflammatory, non-immunogenic and devoid of otherundesired reactions at the administration site. Examples ofpharmaceutically acceptable carriers include, for example, saline,aerosols, commercially available inert gels, or liquids supplementedwith albumin, methyl cellulose or a collagen matrix. Standardpharmaceutical formulation techniques can be employed, such as thosedescribed in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa.

For indications such as bone growth, cartilage repair, cardiac repairand inhibition of restenosis, it may be advantageous to administer thethrombin peptide derivative in a sustained release formulation. Polymersare often used to form sustained release formulations. Examples of thesepolymers include poly α-hydroxy esters such as polylacticacid/polyglycolic acid homopolymers and copolymers, polyphosphazenes(PPHOS), polyanhydrides and poly(propylene fumarates).

Polylactic acid/polyglycolic acid (PLGA) homo and copolymers are wellknown in the art as sustained release vehicles. The rate of release canbe adjusted by the skilled artisan by variation of polylactic acid topolyglycolic acid ratio and the molecular weight of the polymer (seeAnderson, et al., Adv. Drug Deliv. Rev. 28: 5 (1997), the entireteachings of which are incorporated herein by reference). Theincorporation of poly(ethylene glycol) into the polymer as a blend toform microparticle carriers allows further alteration of the releaseprofile of the active ingredient (see Cleek et al., J. Control Release48: 259 (1997), the entire teachings of which are incorporated herein byreference). Ceramics such as calcium phosphate and hydroxyapatite canalso be incorporated into the formulation to improve mechanicalqualities.

PPHOS polymers contain alternating nitrogen and phosphorous with nocarbon in the polymer backbone, as shown below in Structural Formula(II):

The properties of the polymer can be adjusted by suitable variation ofside groups R and R′ that are bonded to the polymer backbone. Forexample, the degradation of and drug release by PPHOS can be controlledby varying the amount of hydrolytically unstable side groups. Withgreater incorporation of either imidazolyl or ethylglycol substitutedPPHOS, for example, an increase in degradation rate is observed (seeLaurencin et al., J. Biomed Mater. Res. 27: 963 (1993), the entireteachings of which are incorporated herein by reference), therebyincreasing the rate of drug release.

Polyanhydrides, shown in Structural Formula (III), have well defineddegradation and release characteristics that can be controlled byincluding varying amounts of hydrophobic or hydrophilic monomers such assebacic acid and 1,3-bis(p-carboxyphenoxy)propane (see Leong et al., J.Biomed. Mater. Res. 19: 941 (1985), the entire teachings of which areincorporated herein by reference). To improve mechanical strength,anhydrides are often copolymerized with imides to formpolyanhydride-co-imides. Examples of polyanhydride-co-imides that aresuitable for orthopaedic applications arepoly(trimellitylimido-glycine-co-1,6-bis(carboxyphenoxy)hexane andpyromellityimidoalanine: 1,6-bis(p-carboxyphenoxy)hexane copolymers.

Carriers for stimulating bone or cartilage growth advantageously includeporous matrices which can then serve as a scaffolding for bone andtissue growth onto which bone progenitor cells and osteogenic cells maymigrate and attach. Such carriers are said to be osteoconductive. Forcertain applications, the carrier should have sufficient mechanicalstrength to maintain its three dimensional structure and help supportthe immobilization of the bone or tissue segments being united orgrafted together.

Examples of suitable osteoconductive carriers include collagen (e.g.,bovine collagen), fibrin, calcium phosphate ceramics (e.g.,hydroxyapatite and tricalcium phosphate), calcium sulfate,guanidine-extracted allogenic bone and combinations thereof. A number ofsuitable carriers are commercially available, such as COLLAGRAFT®((Cohension Technologies, Inc., Palo Alto, Calif.), which is a mixtureof hydroxyapatite, tricalcium phosphate and fibrillar collagen, and PROOSTEON 500™ (Interpore Cross International, Irvine, Calif.), which is ahydroxyapatite biomatrix formed by the conversion of marine coralcalcium carbonate to crystalline hydroxyapatite.

Descriptions of synthetic biodegradable polymers that can serve asosteoconductive carriers with sustained release characteristics, can befound in Behravesh et al., Clinical Orthopaedics 367: S118 (1999) andLichun et al., Polymeric Delivery Vehicles for Bone Growth Factors in“Controlled Drug Delivery—Designing Technologies for the Future” Parkand Mrsny eds., American Chemical Society, Washington, D.C. (2000). Theentire teachings of these references are incorporated herein byreference. Examples of these polymers include poly α-hydroxy esters suchas polylactic acid/polyglycolic acid homopolymers and copolymers,polyphosphazenes (PPHOS), polyanhydrides and poly(propylene fumarates),which are described above in detail.

Implantable pharmaceutical compositions of the present invention areparticularly useful because they can be administered at a site in needof bone growth. “Implantation” or “administration at a site” means insufficient proximity to the site in need of treatment so that bonegrowth occurs (e.g., more bone growth in the presence of the drug thanin its absence) at the site when the thrombin peptide derivative dimeris released from the pharmaceutical composition. These pharmaceuticalcompositions can be shaped as desired in anticipation of surgery orshaped by the physician or technician during surgery. It is preferred toshape the matrix to span a tissue defect and to take the desired form ofthe new tissue. In the case of bone repair of a non-union defect, forexample, it is desirable to use dimensions that span the non-union. Inbone formation procedures, the material is slowly absorbed by the bodyand is replaced by bone in the shape of or very nearly the shape of theimplant. Alternatively, the pharmaceutical compositions can beadministered to the site in the form of microparticles or microspheres.The microparticles are placed in contact or in close proximity to thesite in need of osteoconduction either by surgically exposing the siteand applying the microparticles on or in close proximity to the site bypainting, pipetting, spraying, injecting or the like. Microparticles canalso be delivered to the site by endoscopy or by laparoscopy.

Poly(propylene fumarates) (PPF) are highly desirable biocompatibleimplantable carriers for use in repairing bone defects because they arean injectable, in situ polymerizable, biodegradable material.“Injectable” means that the material can be injected by syringe througha standard needle used for injecting pastes and gels. PPF, combined witha vinyl monomer (N-vinyl pyrrolidinone) and an initiator (benzoylperoxide), forms an injectable solution that can be polymerized in situ.It is particularly suited for filling skeletal defects of a wide varietyof sizes and shapes (see Suggs et al., Macromolecules 30: 4318 (1997),Peter et al., J. Biomater. Sci. Poly,. Ed. 10: 363 (1999) and Yaszemskiet al., Tissue Eng. 1: 41 (1995), the entire teachings of which areincorporated herein by reference). The addition of solid phasecomponents such as tricalcium phosphate and sodium chloride can improvethe mechanical properties of PPF polymers (see Peter et al., J. Biomed.Mater. Res. 44: 314 (1999), the entire teachings of which areincorporated herein by reference).

In yet another alternative, the pharmaceutical composition can bepartially enclosed in a supporting physical structure such as a mesh,wire matrix, stainless steel cage, threaded interbody fusion cage andthe like before administering to the site in need of bone growth.

Injectable delivery formulations may be administered intravenously ordirectly at the site in need of treatment. The injectable carrier may bea viscous solution or gel.

Delivery formulations include physiological saline, bacteriostaticsaline (saline containing about 0.9% mg/mL benzyl alcohol),phosphate-buffered saline, Hank's solution, Ringer's-lactate, or liquidssupplemented with albumin, methyl cellulose, or hyaluronic acid.Injectable matrices include polymers of poly(ethylene oxide) andcopolymers of ethylene and propylene oxide (see Cao et al., J. Biomater.Sci 9: 475 (1998) and Sims et al., Plast Reconstr. Surg. 98: 843 (1996),the entire teachings of which are incorporated herein by reference).

Other compositions which are injectable matrices include the solutionsof poly(propylene fumarate) copolymers described above and pastes ofcalcium phosphate ceramics (see Schmitz et al., J. Oral MaxillofacialSurgery 57: 1122 (1999), the entire teachings of which are incorporatedherein by reference). Injectable matrices can be injected directly tothe site in need of bone growth and can conveniently be used to fillvoids and fuse bones without the need for invasive surgery.

Methods for encapsulating compositions (such as in a coating of hardgelatin or cyclodextran) are known in the art (Baker, et al.,“Controlled Release of Biological Active Agents”, John Wiley and Sons,1986).

Ointments are typically prepared using an oleaginous base, e.g.,containing fixed oils or hydrocarbons, such as white petrolatum ormineral oil, or an absorbent base, e.g., consisting of an absorbentanhydrous substance or substances, for example anhydrous lanolin.Following formation of the base, the active ingredients are added in thedesired concentration.

Creams generally comprise an oil phase (internal phase) containingtypically fixed oils, hydrocarbons, and the like, such as waxes,petrolatum, mineral oil, and the like, and an aqueous phase (continuousphase), comprising water and any water-soluble substances, such as addedsalts. The two phases are stabilized by use of an emulsifying agent, forexample, a surface active agent, such as sodium lauryl sulfate;hydrophilic colloids, such as acacia colloidal clays, beegum, and thelike. Upon formation of the emulsion, the active ingredients are addedin the desired concentration.

Gels are comprised of a base selected from an oleaginous base, water, oran emulsion-suspension base, as previously described. To the base isadded a gelling agent which forms a matrix in the base, increasing itsviscosity to a semisolid consistency. Examples of gelling agents arehydroxypropyl cellulose, acrylic acid polymers, and the like. The activeingredients are added to the formulation at the desired concentration ata point preceding addition of the gelling agent.

Diseases and conditions, treatable with thrombin peptide derivativedimers, for example, wounds and angioplasty, are often accompanied bysymptoms and infirmities such as pain and infection. In certaininstances it may be advantageous to co-administer one or more additionalpharmacologically active agents along with a thrombin peptide derivativedimer to address such issues. For example, managing pain andinflamation, may require co-administration with analgesic or ananti-inflammatory agents. Managing infection may requireco-administration with antimicrobial, antibiotic or disinfectant agents.

Thrombin peptide derivatives can be synthesized by solid phase peptidesynthesis (e.g., BOC or FMOC) method, by solution phase synthesis, or byother suitable techniques including combinations of the foregoingmethods. The BOC and FMOC methods, which are established and widelyused, are described in Merrifield, J. Am. Chem. Soc. 88: 2149 (1963);Meienhofer, Hormonal Proteins and Peptides, C. H. Li, Ed., AcademicPress, 1983, pp. 48-267; and Barany and Merrifield, in The Peptides, E.Gross and J. Meienhofer, Eds., Academic Press, New York, 1980, pp.3-285. Methods of solid phase peptide synthesis are described inMerrifield, R. B., Science, 232: 341 (1986); Carpino, L. A. and Han, G.Y., J. Org. Chem., 37: 3404 (1972); and Gauspohl, H. et al., Synthesis,5: 315 (1992)). The teachings of these six articles are incorporatedherein by reference in their entirety.

Thrombin peptide derivative dimers can be prepared by oxidation of themonomer. Thrombin peptide derivative dimers can be prepared by reactingthe thrombin peptide derivative with an excess of oxidizing agent. Awell-known suitable oxidizing agent is iodine. Specific conditions areprovided in Examples 1 and 2.

The invention is illustrated by the following examples which are notintended to be limiting in any way.

EXEMPLIFICATION Example 1 Formation of Thrombin Peptide Dimer

TP508 was dissolved in a solution of a six parts acetic acid and onepart water. A ten-fold molar excess of iodine was added and the reactionis allowed to proceed with stirring for 90 minutes at room temperature.Excess iodine was removed by extraction with CCl₄ (3 to 4 times). Thedimerized peptide was purified by HPLC on a C18 reverse phase column toremove un-reacted monomers.

Example 2 TP508-Dimer

Conversion of TP508 to Dimer over Time

TP508 was dissolved in saline (sterile 0.9% sodium chloride injectiblesolution) at 5 mg/mL and incubated at 4° C. Over a time period of 6months, triplicate samples were taken at intervals from the solution.The samples were analyzed by HPLC to separate TP508-monomer, TP508-dimerand unknowns. The peak area of TP508-dimer, after three months, showedno decrease over time. No increase in the unknown peaks was observed.The results of FIG. 1 show that TP508-dimer does not revert to monomerover time.

The area percent of each HPLC peak was plotted in FIG. 1. The peak areapercent corresponds directly to the percent of material in solution. Thepeak area of TP508-monomer decreased over time whereas the peak area ofTP508-dimer increased over time. No increase in the unknown peaks wereobserved. The results of FIG. 1 show that TP508 converts to dimer overtime.

TP508-Dimer does not Revert to Monomer over Time

The peak area of TP508-dimer, after three months, showed no decreaseover time. No increase in unknown peaks was observed. The results ofFIG. 1 show that TP508-dimer does not revert to monomer over time.

Example 3 Wound Healing Activity of the Thrombin Peptide Dimer

Methodology and Study Design

The objective of this study was to evaluate the wound healing activityof the dimerized form of the thrombin peptide TP508. The study assessedthe effect of the thrombin peptide dimer on wound closure.

Two, full-thickness, 2 cm-diameter excisions were created on the dorsumof male Sprague-Dawley rats. Both wounds on a given rat were togethertreated with either vehicle with a low-dose of thrombin peptide dimer,vehicle with a high-dose of thrombin peptide dimer, vehicle alone(negative control), or vehicle with TP508 (positive control), yielding atotal of four treatment groups. Each group contained 6 rats. Theactivities of the high and low dose of thrombin peptide dimer werecompared to vehicle alone and to TP508. Wound size was determined onpost-wounding days 3, 7, and 10 by tracing the perimeter of the woundonto an acetate sheet and using digital analysis to compute the surfacearea of each wound.

Preparation of Treatment Solutions

Saline Solution

D-mannitol (20 mg) was dissolved in 12.5 mL of saline (sterile 0.9%sodium chloride injectible solution) to yield a solution of 8.9 mMD-mannitol in saline. This solution was used as the vehicle control forthis experiment.

TP508 Solution

Lyophilized TP508 (1 mg) was dissolved in 1 mL of the D-mannitol/salinevehicle. The stock solution (1 mg/mL) was further diluted in vehicle toyield a working solution of 2.5 μg/mL. The working solution wasmaintained on ice throughout the experiment.

Thrombin Peptide Dimer Solution

High-Dose

TP508 (12.5 μg) and 2 mg D-mannitol were dissolved in 1.25 mL of saline(without D-mannitol) to yield a stock solution of 10 μg TP508 per mL of8.9 mM D-mannitol in saline. The stock solution was used directly as thehigh treatment dose (0.4 μg per 40 μL per wound). The stock solution wasmaintained on ice throughout the experiment.

Low-Dose

The low treatment dose was prepared by further dilution of the stockdimer solution in the D-mannitol/saline vehicle to obtain a workingsolution of 2.5 μg per mL (0.1 μg per 40 μL per wound). The workingsolution was maintained on ice throughout the experiment.

Wound Treatment

Both wounds on a given animal received the same treatment: a single,topical application of a 40 μL volume containing vehicle alone, vehiclewith TP508 (0.1 μg/mL), or vehicle with thrombin peptide dimer (0.1μg/mL or 0.4 μg/mL).

Observations and Wound Size Analysis

The rats were observed for ten days following wounding, and no clinicalsigns of abnormal behavior, infection or toxicity were noted. Onpost-wounding days 3, 7, and 10, the wounds were evaluated by tracingthe wound perimeter onto a flexible acetate sheet, then determiningwound area with digital analysis software.

The results of the experiment are presented in FIG. 2. FIG. 2 showswound area measurements from post-wounding Day 7 and Day 10. Nodifferences in wound size between the groups were present onpost-wounding Day 3. Each data point represents the mean and standarderror of the mean of 12 wounds from 6 rats. Statistical comparisonsbetween groups were made using a repeated measures analysis of variance;Fisher's LSD was used for post hoc testing between groups.

In this experiment, both TP508 and thrombin peptide dimer producedsignificantly smaller wounds than vehicle alone by post-wounding Day 7.TP508-treated wounds were 21.3% smaller in area than vehicle-controls,while wounds treated with the same dose of thrombin peptide dimer (0.1μg per wound) were 29.2% smaller in area than vehicle-controls. Thedifference between TP508 and thrombin peptide dimer treatments was notstatistically significant. On day 10, as on Day 7, TP508 and both dosesof thrombin peptide dimer generated wounds that were significantlysmaller than those of the controls. In addition, a statisticallysignificant difference was found on Day 10 between the TP508 treatedgroup and the groups treated with the thrombin peptide dimer (p<0.05).On day 10, TP508 treated wounds were 23.1% smaller in area thanvehicle-controls, while wounds treated with the same dose of thrombinpeptide dimer (0.1 μg per wound) were 41.4% smaller in area thanvehicle-controls.

In the low dose group, the thrombin peptide dimer was administered at anequivalent weight to the TP508 group. That is, each of these groupsreceived 0.1 μg of peptide per wound, resulting in half as many moles ofdimer as monomer administered to wounds in these two groups.

The results indicated that the thrombin peptide dimer is biologicallyactive in accelerating wound closure. When viewed as “wound closure”,the thrombin peptide dimer generated a statistically significant effecton healing that was equivalent to the effect of TP508.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A peptide dimer comprising two thrombin peptide derivatives which,independently, comprise a polypeptide consisting of the amino acidsequence of SEQ ID NO. 2(Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or aC-terminal truncated fragment thereof having at least six amino acids,provided that zero, one, two, or three amino acids in the polypeptidediffer from the corresponding position of SEQ ID NO. 2; said thrombinpeptide derivatives optionally comprising a C-terminal amide; and saidthrombin peptide derivatives optionally comprising an acylatedN-terminus.
 2. The dimer of claim 1, wherein the dimer is essentiallyfree of monomer.
 3. The dimer of claim 2, wherein the thrombin peptidederivatives are the same.
 4. The dimer of claim 3, wherein the thrombinpeptide derivatives are covalently linked through a disulfide bond. 5.The dimer of claim 4, wherein the thrombin peptide derivatives consistof between about 12 and about 23 amino acids.
 6. The dimer of claim 5,wherein the thrombin peptide derivatives comprise a C-terminal amide andoptionally comprise an acylated N-terminus, wherein said C-terminalamide is represented by —C(O)NR_(b)R_(c), wherein R_(b) and R_(c) areindependently hydrogen, a C₁-C₁₀ substituted or unsubstituted aliphaticgroup, or R_(b) and R_(c), taken together with the nitrogen to whichthey are bonded, form a C₁-C₁₀ non-aromatic heterocyclic group, and saidN-terminal acyl group is represented by R_(d)C(O)—, wherein R_(d) ishydrogen, a C₁-C₁₀ substituted or unsubstituted aliphatic group, or aC₁-C₁₀ substituted or unsubstituted aromatic group.
 7. The dimer ofclaim 6, wherein the thrombin peptide derivatives comprise an N-terminuswhich is unsubstituted and a C-terminus which is unsubstituted or aC-terminal amide represented by —C(O)NH₂.
 8. The dimer of claim 4,wherein the thrombin peptide derivatives consist of between about 12 andabout 33 amino acids.
 9. The dimer of claim 8, wherein the thrombinpeptide derivatives comprise a C-terminal amide and optionally comprisean acylated N-terminus, wherein said C-terminal amide is represented by—C(O)NR_(b)R_(c), wherein R_(b) and R_(c) are independently hydrogen, aC₁-C₁₀ substituted or unsubstituted aliphatic group, or R_(b) and R_(c),taken together with the nitrogen to which they are bonded, form a C₁-C₁₀non-aromatic heterocyclic group, and said N-terminal acyl group isrepresented by R_(d)C(O)—, wherein R_(d) is hydrogen, a C₁-C₁₀substituted or unsubstituted aliphatic group, or a C₁-C₁₀ substituted orunsubstituted aromatic group.
 10. The dimer of claim 9, wherein thethrombin peptide derivatives comprise an N-terminus which isunsubstituted and a C-terminus which is unsubstituted or a C-terminalamide represented by —C(O)NH₂.
 11. The dimer of claim 7, wherein thethrombin peptide derivatives comprise a polypeptide consisting of theamino acid sequence of SEQ ID NO. 2(Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val), or aC-terminal truncated fragment thereof having at least six amino acids,provided that zero, one or two of the amino acids in the thrombinpeptide derivatives are conservative substitutions of the correspondingamino acid in SEQ ID NO
 2. 12. The dimer of claim 7, wherein thethrombin peptide derivatives comprise a polypeptide consisting of theamino acid sequence of SEQ ID NO 4(Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val, wherein X₁ isGlu or Gln and X₂ is Phe, Met, Leu, His or Val).
 13. The dimer of claim7, wherein the thrombin peptide derivatives have the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO 5), or a fragment thereof comprising amino acids 10-18 of SEQID NO 5, provided that zero, one, two or three amino acids in thethrombin peptide derivatives differ from the amino acid at thecorresponding position of SEQ ID NO
 5. 14. The dimer of claim 7, whereinthe thrombin peptide derivatives have the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO 5), or a fragment thereof comprising amino acids 10-18 of SEQID NO 5, provided that zero, one or two amino acids in the thrombinpeptide derivatives are conservative substitutions of the amino acid atthe corresponding position of SEQ ID NO
 5. 15. The dimer of claim 7,wherein the thrombin peptide derivatives have the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val(SEQ ID NO. 6), wherein X₁ is Glu or Gln and X₂ is Phe, Met, Leu, His orVal) or a fragment thereof comprising amino acids 10-18 of SEQ ID NO. 6.16. The dimer of claim 7, wherein the thrombin peptide derivatives havethe amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val(SEQ ID NO. 6), wherein X₁ is Glu or Gln and X₂ is Phe, Met, Leu, His orVal.
 17. The dimer of claim 16, wherein X₁ is Glu and X₂ is Phe.
 18. Thedimer of claim 7, wherein the thrombin peptide derivatives have theamino acid sequenceH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-NH₂(SEQ ID NO. 11), wherein X₁ is Glu or Gln and X₂ is Phe, Met, Leu, Hisor Val.
 19. The dimer of claim 18, wherein X₁ is Glu and X₂ is Phe. 20.A peptide dimer having two thrombin derivatives, each with the aminoacid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 3), wherein the thrombin peptide derivatives are covalentlylinked by a disulfide bond; said thrombin peptide derivatives optionallycomprising a C-terminal amide; and said thrombin peptide derivativesoptionally comprising an acylated N-terminus.
 21. A peptide dimerrepresented by the following structural formula:


22. The dimer of claim 10, wherein the thrombin peptide derivatives havethe amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr(SEQ ID NO. 7), or a C-terminal truncated fragment thereof having atleast twenty-three amino acids, provided that zero, one, two three aminoacids in the thrombin peptide derivatives differ from the correspondingamino acid in SEQ ID NO.
 9. 23. The dimer of claim 10, wherein thethrombin peptide derivatives have the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr(SEQ ID NO. 7), or a C-terminal truncated fragment thereof having atleast twenty-three amino acids, provided that zero, one or two of theamino acids in the thrombin peptide derivatives are conservativesubstitutions of the corresponding amino acid in SEQ ID NO.
 9. 24. Thedimer of claim 10, wherein the thrombin peptide derivatives have theamino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr(SEQ ID NO. 7), or a C-terminal truncated fragment thereof having atleast twenty-three amino acids.
 25. A method of treating a subject inneed of treatment with a thrombin receptor agonist, said methodcomprising the step of administering an effective amount of a peptidedimer comprising two thrombin peptide derivatives which, independently,comprise a polypeptide consisting of the amino acid sequence of SEQ IDNO. 2 (Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val) or aC-terminal truncated fragment thereof having at least six amino acids,provided that zero, one, two, or three amino acids in the thrombinpeptide derivative differ from the corresponding position of SEQ ID NO.2; said thrombin peptide derivatives optionally comprising a C-terminalamide; and said thrombin peptide derivatives, optionally comprising anacylated N-terminus.
 26. The method of claim 25, wherein the subject isin need of treatment to promote cardiac repair.
 27. The method of claim25, wherein the subject is in need of treatment to promote cartilagegrowth or repair.
 28. The method of claim 25, wherein the subject is inneed of bone growth.
 29. The method of claim 25, wherein the site is inneed of a bone graft.
 30. The method of claim 28, wherein the site is asimple fracture, segmental gap in a bone, a bone void or at a non-unionfracture.
 31. The method of claim 25, wherein the subject is in need oftreatment to promote wound healing.
 32. The method of claim 25, whereinthe subject is in need of treatment to inhibit restenosis.
 33. Themethod of claim 25, wherein the dimer is essentially free of monomer.34. The method of claim 25, wherein the thrombin peptide derivatives arethe same.
 35. The method of claim 25, wherein the thrombin peptidederivatives are covalently linked through a disulfide bond.
 36. Themethod of claim 25, wherein the thrombin peptide derivatives consist ofbetween about 12 and about 23 amino acids.
 37. The method of claim 36,wherein the thrombin peptide derivatives wherein comprise a C-terminalamide and optionally comprise an acylated N-terminus, said C-terminalamide is represented by —C(O)NR_(b)R_(c), wherein R_(b) and R_(c) areindependently hydrogen, a C₁-C₁₀ substituted or unsubstituted aliphaticgroup, or R_(b) and R_(c), taken together with the nitrogen to whichthey are bonded, form a C₁-C₁₀ non-aromatic heterocyclic group, and saidN-terminal acyl group is represented by R_(d)C(O)—, wherein R_(d) ishydrogen, a C₁-C₁₀ substituted or unsubstituted aliphatic group, or aC₁-C₁₀ substituted or unsubstituted aromatic group.
 38. The method ofclaim 37, wherein the thrombin peptide derivatives comprise anN-terminus which is unsubstituted and a C-terminus which isunsubstituted or a C-terminal amide represented by —C(O)NH₂.
 39. Themethod of claim 25, wherein the thrombin peptide derivatives consist ofbetween about 12 and about 33 amino acids.
 40. The method of claim 39,wherein the thrombin peptide derivatives comprise a C-terminal amide andoptionally comprise an acylated N-terminus, wherein said C-terminalamide is represented by —C(O)NR_(b)R_(c), wherein R_(b) and R_(c) areindependently hydrogen, a C₁-C₁₀ substituted or unsubstituted aliphaticgroup, or R_(b) and R_(c), taken together with the nitrogen to whichthey are bonded, form a C₁-C₁₀ non-aromatic heterocyclic group, and saidN-terminal an acyl group is represented by R_(d)C(O)—, wherein R_(d) ishydrogen, a C₁-C₁₀ substituted or unsubstituted aliphatic group, or aC₁-C₁₀ substituted or unsubstituted aromatic group.
 41. The method ofclaim 40, wherein the thrombin peptide derivatives comprise anN-terminus which is unsubstituted and a C-terminus which isunsubstituted or a C-terminal amide represented by —C(O)NH₂.
 42. Themethod of claim 38, wherein the thrombin peptide derivatives comprise apolypeptide consisting of the amino acid sequence of SEQ ID NO. 2(Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val), or aC-terminal truncated fragment thereof having at least six amino acids,provided that zero, one or two of the amino acids in the thrombinpeptide derivatives are conservative substitutions of the correspondingamino acid in SEQ ID NO
 2. 43. The method of claim 38, wherein thethrombin peptide derivatives comprise a polypeptide consisting of theamino acid sequence of SEQ ID NO 4(Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val, wherein X₁ isGlu or Gln and X₂ is Phe, Met, Leu, His or Val).
 44. The method of claim38, wherein the thrombin peptide derivatives have the amino acidsequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO 5), or a fragment thereof comprising amino acids 10-18 of SEQID NO 5, provided that zero, one, two or three amino acids in thethrombin peptide derivatives differ from the amino acid at thecorresponding position of SEQ ID NO
 5. 45. The method of claim 38,wherein the thrombin peptide derivatives have the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 5), or a fragment thereof comprising amino acids 10-18 ofSEQ ID NO. 5, provided that zero, one or two amino acids in the thrombinpeptide derivatives are conservative substitutions of the amino acid atthe corresponding position of SEQ ID NO.
 5. 46. The method of claim 38,wherein the thrombin peptide derivatives have the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val(SEQ ID NO. 6), wherein X₁ is Glu or Gin and X₂ is Phe, Met, Leu, His orVal) or a fragment thereof comprising amino acids 10-18 of SEQ ID NO. 6.47. The method of claim 38, wherein the thrombin peptide derivativeshave the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val(SEQ ID NO. 6), wherein X₁ is Glu or Gin and X₂ is Phe, Met, Leu, His orVal.
 48. The method of claim 47, wherein X₁ is Glu and X₂ is Phe. 49.The method of claim 38, wherein the thrombin peptide derivatives havethe amino acid sequenceH-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-NH₂(SEQ ID NO. 11), wherein X₁ is Glu or Gln and X₂ is Phe, Met, Leu, Hisor Val.
 50. The method of claim 49, wherein X₁ is Glu and X₂ is Phe. 51.A method of treating a subject in need of treatment with a thrombinreceptor agonist, said method comprising the step of administering aneffective amount of a peptide dimer having two thrombin peptidederivatives with the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val(SEQ ID NO. 3), wherein the thrombin peptide derivatives are covalentlylinked by a disulfide bond; said thrombin peptide derivatives optionallycomprising a C-terminal amide; and said thrombin peptide derivativesoptionally comprising an acylated N-terminus.
 52. A method of treating asubject in need of treatment with a thrombin receptor agonist, saidmethod comprising the step of administering an effective amount ofpeptide dimer represented by the following structural formula:


53. The method of claim 41, wherein the thrombin peptide derivativeshave the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr(SEQ ID NO. 7), or a C-terminal truncated fragment thereof having atleast twenty-three amino acids, provided that zero, one, two three aminoacids in the thrombin peptide derivatives differ from the correspondingamino acid in SEQ ID NO.
 9. 54. The method of claim 41, wherein thethrombin peptide derivatives have the amino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr(SEQ ID NO. 7), or a C-terminal truncated fragment thereof having atleast twenty-three amino acids, provided that zero, one or two of theamino acids in the thrombin peptide derivatives are conservativesubstitutions of the corresponding amino acid in SEQ ID NO.
 7. 55. Themethod of claim 41, wherein the thrombin peptide derivatives have theamino acid sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-Asn-Asn-Arg-Trp-Tyr(SEQ ID NO. 7), or a C-terminal truncated fragment thereof having atleast twenty-three amino acids.